Objective: This phase Ia study was designed to assess the pharmacokinetic (PK) characters of free vincristine  and total vincristine , urinary excretion and safety of intravenous administration of vincristine sulfate liposomes injection (VSLI) in Chinese patients with malignant lymphoma and compare the results with those for conventional vincristine sulfate injection (VSI).Methods: In the phase Ia, randomized, open-label, two sequence cross-over study, patients from one group were exposed to treatment 1 including cytoxan (cyclophosphamide power injection), hydroxyrubicin (adriamycin power injection), oncovin (VSI), and prednisone tablets (standard CHOP scheme) before crossed over to treatment 2 (modified CHOP scheme in which VSI was replaced with VSLI). Patients from another group received treatments in reverse order.Results: In this phase Ia study, a total of eight subjects participated. VCR elimination from the circulation after injection of VSLI was characterized by a significantly increased maximum concentration  and plasma area under the plasma concentration-time curve from zero to infinity , markedly decreased distribution volume  and plasma clearance  compared to lower Cmax (26.6 ng/mL) and AUC0-Inf (95.1 ng/mL h), larger Vz (688.8 L) and CL (22.1 L/h) for VSI. The small proportion of F-VCR following infusion of VSLI in circulation was reflected by very low Cmax (1.8 ng/mL) and AUC0-Inf (50.5 ng/mL h). Less than 3% of the administered dose of VSLI was excreted in urine and the extent was similar to that for VSI. The elimination percentage of 40–21–14% for VSI changed to 6.2–24–39% for VSLI at intervals of 0–5, 5–13 and 13–25 h, respectively. Significant difference of toxicity between VSLI and VSI was not observed.Conclusion: VSLI exhibits higher AUC0-Inf of T-VCR, lower CL and Vz compared with VSI. VSLI was well tolerated, maybe due to the markedly decreasing AUC0-Inf of F-VCR. The majority of VCR was enveloped in liposome and VCR was released gradually from liposome following injection of VSLI. Liposomal encapsulation of VCR does not alter the route and extent of VCR excretion in urine. After the standard procedure, the encapsulation efficiency of liposomal VCR was up to 95% and liposomal VCR remains stable for at least 24 h when stored at 4°C. VSI, cytoxan (cyclophosphamide power injection), hydroxyrubicin (adriamycin power injection), oncovin (VSI) and prednisone tablets were purchased through Nanjing Pharmaceutical Hefei Tianxing Co., Ltd., China.9/L, an absolute granulocyte count ≥1.5 × 109/L, a platelet count 75.0 × 109/L and a hemoglobin count ≥80g/L), and adequate hepatic function , and renal function (serum creatinine ≤1.5 × ULN).Eligible patients aged from 18 to 65 years had histologically or cytologically confirmed malignant lymphoma, which were refractory to conventional forms of cancer therapy. They should not receive other therapy including surgery, chemotherapy or radiotherapy within at least previous 4 weeks. In addition, it was required for eligible patients to have life expectancy of at least 12 weeks, and Eastern Cooperative Oncology Group performance status of 0 to 2, and adequate bone marrow function , hydroxyrubicin , oncovin  and prednisone tablets (100 mg). The drugs for injection were administrated intravenously (I.V.) on study day 1 and the tablets were swallowed whole for concessive 5 days from day 1 to 5 of each 21-day circle. Treatment 2 was modified CHOP scheme in which VSLI (1.0 mg/m2) replaced VSI and others remained unchanged. VSI and VSLI should be administrated within 60 ± 5 min.The study design was shown in Subjects fasted overnight for at least 10 h prior to drug administration and for at least 2 h post-dose, after which controlled food intake was allowed. Cigarette, alcohol, tea, coffee, grapefruit-, or drug-containing food or drink, and non-investigator-approved prescription medications or over-the-counter products were to be avoided at least 24 h post-dose and during the study.g at 4°C for 10 min) and obtained plasma then was divided into three parts. One part was used to separate F-VCR from liposomes using solid phase extraction (SPE) method immediately and the remaining two parts (one part was used as the backup) were frozen for assay of the T-VCR concentration.Blood samples were collected at pre-dosing, 15, 30, and 60 min after infusion and 0.25, 0.5, 2, 4, 8, 12, 24, 48, 72, and 96 h after the end of infusion. The whole blood was centrifuged  before and after drug infusion up to 97 h at six intervals: 0–5, 5–13, 13–25, 25–49, 49–73, and 73–97 h. The samples from each interval were mixed and the volume was measured. Then 10 mL urine samples were remained and frozen at -80°C.m/z 413.2 → 353.2 and m/z 406.2 → 271.6, respectively. F-VCR was separated from liposomal form in plasma using SPE method; plasma T-VCR and urine VCR were extracted using liquid-liquid extraction method. F-VCR and T-VCR were identified and quantified over a theoretical concentration range of 0.2–50 ng/ml for F-VCR in plasma, 0.5–400 ng/ml for T-VCR in plasma and 0.5–100 ng/ml for VCR in urine, respectively. Assay specificity was assessed using blank sample from six different lots to verify the absence of interference at retention time. Quantitation was made using peak area ratios of analyte/IS, and back-calculated concentrations were determined using a weighted (1/ × 2) linear regression (y = ax + b).The plasma concentration of F-VCR and T-VCR and the urine VCR concentration was determined using the ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) system  with vinblastine sulfate used as the internal standard (IS) as previously described by our team , 2015. Tmax and the time to reach Cmax (Tmax) directly. The main PK parameters, calculated from the plasma concentration-time data using a non-compartmental analysis method , were elimination half-life (t1/2), the area under plasma concentration-time curve from zero to last time (AUC0-t) and from zero to infinity (AUC0-Inf), the mean retain time from zero to last time (MRT0-t) and from zero to infinity (MRT0-Inf), clearance (CL) and apparent volume of distribution (Vd). Mann-Whitney test was used to compare PK parameters  between the two groups using GraphPad Prism software (Version 7.0). P-value less than 0.05 was considered to be significant.The measured plasma concentration was used to obtain CThe safety of the subjects was monitored by evaluation of physical examinations, vital signs, electrocardiograms, and clinical laboratory tests and adverse events (AEs) reporting. AEs were classified by system organ class and were graded according to National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE), version 4. Safety results were analyzed using descriptive statistics.Table 1. Nobody withdrew/were withdrawn before the end of the study. All patients were assessable for safety. All subjects provided measurable PK data and were therefore included in the PK evaluation and statistical analysis. Table 2 is a summary of dose level, number of the patients and actual dose delivered.There were eight patients  enrolled in this study. Other demographic data are shown in 2) or VSI (1.4 mg/m2) are shown in Figure 2. The corresponding PKs of T-VCR were calculated from these data and are presented in Table 3. The maximum plasma T-VCR concentrations (Cmax) were observed at the end of the VSLI infusion, subsequently a rapid distribution phase occurred, in which concentration rapidly declined within 1 h, followed by a slow distribution phase and a terminal elimination phase. Therefore, the T-VCR plasma concentrations for all subjects were characterized by a triexponential decline after treated with VSLI. Although the profiles for VSI are very similar to those for VSLI, VSI exhibited significant lower Cmax and AUC0-Inf of VCR, as well as markedly higher Vz and CL than those of T-VCR for VSLI. After administration of VSLI, significantly elevated systematic exposure (Cmax was 86.6 ng/mL and AUC0-Inf was 222.1 ng/mL h), associated with decreased CL (8.9 L/h) and Vz (224.2 L) of T-VCR were obtained, compared with those of VCR for VSI (P < 0.01). There was no significant difference for half life (t1/2) between two groups .The mean plasma T-VCR concentration vs. time curves of patients who received VSLI  of F-VCR was obtained at the end of VSLI infusion (1 h) in most patients (7 of 8 patients). Another one or two lower concentration peaks appeared at 9 or 3/9 h after the start of infusion. Then, the F-VCR concentration gradually declined. At 1 h after i.v. administration of VSLI, the mean plasma concentration of F-VCR was 1.8 ng/ml, compared with 86.6 ng/ml for T-VCR. This indicated that F-VCR represented 2% of the T-VCR in the plasma at this time point. The proportion of F-VCR in the plasma was evaluated by comparing the AUC values. The AUC0-Inf value for F-VCR after the administration of VSLI was 64.8 ng/mL h, which represent 29.2% of the AUC0-Inf for T-VCR. In addition, systemic exposure of F-VCR for VSLI was compared with that of VCR for VSI. Although the similar AUC0-Inf value of F-VCR to that of VCR was obtained after dose calibration, the lower F-VCR concentration at every time point from 0.25 to 25 h after administration of VSLI than that of VCR for VSI was observed. It is important to note that the significant decreased Cmax of F-VCR contrasts the increased plasma VCR concentration observed after VSI administration where peak value was 26.3 ng/mL, 16-fold greater than that of F-VCR.Multiple peaks were apparently observed for the individual F-VCR concentration-time curve (2) and VSI (1.4 mg/m2) are shown in Figure 3, respectively. Less than 3% of the injected VSLI dose was eliminated in the urine over the 97-h period in the form of unchanged VCR and the extent of urinary excretion was similar to that after treated with VSI. Notably, though about 70% of the total amount of VCR excreted in the urine was recovered within 25 h after both VSLI and VSI administration, time course of excretion had large difference. The majority of VCR was excreted during the first few hours  post infusion of VSI. In contrast, the VCR excretion rate gradually increased over the 25-h period  after VSLI administration.The cumulative excretion percentage of VCR in urine from four patients treated with VSLI (1.0 mg/mTable 4). Eight patients (100%) experienced AEs both in treatment 1and 2, seven (87.5%) and four (50.0%) of whom experienced grade ≥3 AE in treatment 1 and 2, respectively. Hematologic toxicity were the most frequently reported AEs  followed by metabolic disorder  and gastrointestinal reaction . Eight patients showed good tolerance using treatment 1and 2 and there was no significant difference of AEs between two treatments, showing good safety of VSLI at 1.0 mg/m2 dose level. No serious AEs occurred in this study.Adverse events (AEs) were graded according to NCI CTCAE for Adverse Events (version 4.0). AEs mentioned in this paper refer to AEs associated with VALI or VSI  and liposome-entrapped VCR. It is theoretically agreed that F-VCR is responsible for causing toxicity and liposomal VCR accounts for providing the activity. From the safety perspective, it is essential to ensure that the plasma levels of F-VCR post injection of VSLI are comparable with or below those of VCR after treatment with VSI. Thus, it was anticipated that VCR is released from the liposome to increase the concentration of VCR in tumor site and to decrease the concentration of free drug in plasma, then the vast majority of drug present in circulation remains entrapped with liposomes. In order to confirm this hypothesis and better understand the release character of VCR from liposome of VSLI in human being, we developed an SPE method to separate the F-VCR from the liposomal VCR in plasma and the concentration of F-VCR was determined . Based oIt is expected that the excretion route of VCR released from liposome was similar to that of traditional VCR. The biliary system is the principal excretion route for traditional VCR . To conferved even though the 2.8-times higher plasma AUC0-Inf of T-VCR for VSLI than that of VCR for VSI was obtained.Vincristine sulfate liposome injection was well tolerated, no serious AEs were reported and no patient was withdrawn due to an AE during the study. The profile and frequency of AE, as well as the numbers of the patients who experienced the grade ≥3 AE for VSLI were consistent with that for conventional VSI. No additional toxicity was obsIn summary, the high exposure of T-VCR and low exposure of F-VCR demonstrated that the proportion of F-VCR was very small and most of VCR was entrapped in liposome in circulation following injection of VSLI, which explained the fact that VSLI showed higher efficacy and lower toxicity than that for VSI. VCR was released from liposome slowly, which was reflected by the low plasma concentration of F-VCR and the delayed excretion of VCR in urine. Liposomal encapsulation of VCR did not alter the extent of VCR excretion in urine, but time course changed.Clinical Trials.gov Identifier: 2009L01014) was conducted at three sites in China. It was approved by the Ethics Committee of Peking Union Medical College Hospital , Nanjing First Hospital Affiliated of Nanjing Medical University  and The Second Affiliated Hospital of Soochow University , respectively. The study was conducted in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice guidelines. The written Informed Consent Form was obtained from each subject prior to any study procedure.The phase Ia period (FY and MJ contributed equally to the study design, process of samples, data analysis, and article writing. ML assisted with the process of samples and data acquisition. PH was involved in the design of the study and article writing. JJ and HW were the principal investigators of this trial and were involved in the study design and analysis of data. All authors revised the article critically and gave final approval of the version to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Gallbladder cancer is the most common malignant tumor of the biliary tract, and this condition has a rather dismal prognosis, with an extremely low five-year survival rate. To improve the outcome of unresectable and recurrent gallbladder cancer, it is necessary to develop new effective treatments and drugs. The purpose of the present study was to evaluate the effects of cordycepin on human gallbladder cells and uncover the molecular mechanisms responsible for these effects. The Cell Counting Kit-8 (CCK-8) and colony formation assays revealed that cordycepin affected the viability and proliferation of human gallbladder cancer cells in a dose- and time-dependent manner. Flow cytometric analysis showed that cordycepin induced S phase arrest in human gallbladder cancer cell lines(NOZ and GBC-SD cells). Cordycepin-induced apoptosis was observed using an Annexin V/propidium iodide (PI) double-staining assay, and the mitochondrial membrane potential (ΔΨm) decreased in a dose-dependent manner. Additionally, western blot analysis revealed the upregulation of cleaved-caspase-3, cleaved-caspase-9, cleaved-PARP and Bax and the downregulation of Bcl-2, cyclin A and Cdk-2 in cordycepin-treated cells. Moreover, cordycepin inhibited tumor growth in nude mice bearing NOZ tumors. Our results indicate that this drug may represent an effective treatment for gallbladder carcinoma. Gallbladder cancer, an aggressive and highly lethal malignancy, is the most frequent cancer of the biliary tract and the most common neoplasm of the digestive system ,2,3,4,5.Cordyceps sinensis, is widely used in traditional Chinese medicine [in vitro using purified RNA polymerases and poly(A) polymerases from a number of organisms, including yeast and mammals [Cordycepin (3'-deoxyadenosine) , a chiefmedicine ,11. This mammals . In clin mammals ,17,18,1950 (the concentration at which 50% inhibition of cell growth was achieved) of cordycepin in NOZ and GBC-SD cells at 48 h was approximately 19.2 μg/mL and 398.1 μg/mL, respectively, which indicates that cordycepin could inhibit the proliferation capability of gallbladder cancer cells.To confirm the inhibitory effect of cordycepin on cell proliferation, the CCK-8 assay was used. After treatment with cordycepin at various concentrations  for 24, 48 and 72 h, both NOZ and GBC-SD cells showed a dose- and time-dependent decrease in viability. Growth curves for these experiments are shown in Additionally, we investigated the effect of cordycepin on the proliferation of gallbladder cancer cells using a colony assay. As shown in In the experiment, we found that the effective concentration of cordycepin was much lower for NOZ cells. The main conclusion to draw from this observation is that not all cancer cell lines are equally sensitive to cordycepin. This makes it likely that not all gallbladder tumors will respond to cordycepin, and it is therefore important for the development of cordycepin as a drug to understand what determines cordycepin sensitivity, so that the correct patient group to treat with the drug can be identified. Additionally, we guess that this may be caused by differences in AMP-activated protein kinase (AMPK) expression in these gallbladder cancer cells, and we are planning to perform western blots assay for AMPK gamma isoforms to confirm it. AMPK is an energy-sensing enzyme that maintains the balance between ATP production and consumption in all eukaryotic cells. AMPK can sense cellular energy levels; when the energy status is compromised, the system activates catabolic pathways and switches off protein, carbohydrate and lipid biosynthesis, as well as cell growth and proliferation ,21.0/G1 phase and significantly increased the percentage of NOZ and GBD-SD cells in the S phase. These results indicate that cordycepin arrests the cell cycle at the S phase in a dose-dependent manner and can suppress tumor growth by preventing proper DNA replication.To determine the effect of cordycepin on the cell cycle distribution of gallbladder cancer cells, we performed flow cytometry assays. As shown in Additionally, we investigated the effects of cordycepin on cell cycle-related proteins. After treatment with cordycepin for 48 h, the expression of the cell cycle regulatory proteins, cyclin A and Cdk-2, which represent two key regulators of the S phase , was decTo further evaluate the apoptosis-inducing capability of cordycepin in NOZ and GBC-SD cells, Annexin V-FITC/PI double staining and flow cytometry were conducted. Annexin V-FITC positivity and PI negativity (Q4 quadrant) were considered to represent early apoptotic cells; Annexin V-FITC positivity and PI-positivity (Q2 quadrant) represented late apoptotic cells; and Annexin V-FITC negativity and PI negativity (Q3 quadrant) indicated non-apoptotic cells. After treatment with cordycepin for 48 h, the number of viable cells was reduced, whereas the numbers of both early and late apoptotic cells were significantly increased in a dose-dependent manner. As shown in A variety of key events during apoptosis involve the mitochondria. Moreover, cells cannot gain a growth advantage simply by losing mitochondria, and mitochondria dysfunction is often associated with changes in ΔΨm. To evaluate whether mitochondrial membrane integrity was damaged by treatment with cordycepin, ΔΨm in human gallbladder cells was examined using Rhodamine 123, a yellow-green fluorescent probe that stains mitochondria in living cells in a membrane potential-dependent manner. In To evaluate the molecular mechanism responsible for the apoptotic effect of cordycepin on NOZ and GBC-SD cells, the expression of apoptosis-related proteins  was evaluated by western blot analysis after treatment with various concentrations of cordycepin for 48 h. As shown in Cordyceps sinensis, which is widely used in traditional Chinese medicine, had been shown to possess anticancer effects in a broad range of human cancer cells by regulating the cell cycle and apoptosis [The deregulation of apoptosis serves as an indicator of carcinogenesis , and thepoptosis ,31,32. Hpoptosis . This papoptosis ,35. The poptosis . We founOur study also investigated the caspase family, which consists of cysteine proteases that are indispensable in the execution process of apoptosis. Caspase-9 is activated in the mitochondria-mediated intrinsic pathway, and caspases-3 is a key regulator in the caspase-dependent cell apoptosis pathway. In addition, the activation of caspase-3 leads to the final destruction of a target cell . A numbein vivo, as treatment with cordycepin was shown to suppress the growth of human gallbladder cells in vitro. Our results indicated that the tumor growth in nude mice injected with NOZ tumor cells was significantly reduced in a dose-dependent manner without any loss of bodyweight or systemic toxicity after treatment with cordycepin compared with mice injected with PBS vehicle alone (p < 0.05). Thus, our results show that cordycepin could effectively suppress tumor growth in vivo.It was reported that cordycepin has no effect on bodyweight loss or systemic toxicity through intragastric or intraperitoneal administration at the maximal tolerant dose in mice ,39. Therle alone  , antibiotics, fetal bovine serum and trypsin were purchased from Gibco . Cordycepin was obtained from the National Institute for the Control of Pharmaceutical and Biological Products . An Annexin V/Dead Cell Apoptosis Kit was purchased from Invitrogen . The primary antibodies used for western blotting were as follows: rabbit anti-Bcl-2, anti-Bax, anti-caspase-3, anti-caspase-9, anti-PARP, anti-cleaved-caspase-3, anti-cleaved-caspase-9, anti-cleaved-PARP, anti-Cdk-2, anti-Cyclin A and mouse anti-β-actin. All of the antibodies were purchased from Cell Signaling Technology .2. GBC-SD cells were cultured in high-glucose DMEM  supplemented with 100 U/mL penicillin-streptomycin and 10% fetal bovine serum at 37 °C in an incubator containing 5% CO2.The human gallbladder cancer cell line NOZ was purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences . The human gallbladder cancer cell line GBC-SD was purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (CAS). NOZ cells were cultured in William’s medium  supplemented with 100 U/mL penicillin-streptomycin  and 10% fetal bovine serum  in a humidified incubator at 37 °C and 5% CO3 cells per well and incubated overnight. Then, the cells were treated with cordycepin at different concentrations . Cell viability was quantified using a Cell Counting Kit-8  at 24, 48 and 72 h after culture with cordycepin, as previously described. The optical density was measured at 450 nm using a microtiter plate reader , and the cell survival rate was expressed as the absorbance relative to that of untreated controls. The results represent the average of three independent experiments conducted over multiple days.NOZ and GBC-SD cells were seeded into 96-well plates at a density of 4 × 10Cells in a logarithmic growth phase were trypsinized into single-cell suspensions and plated in a 6-well plate at a density of 400 cells per well. After adherence, NOZ and GBC-SD cells were treated with cordycepin for 48 h . Cells were cultured for approximately 14 days, fixed with 4% paraformaldehyde and stained with 0.1% crystal violet . After washing, plates were air-dried, and stained colonies were photographed using a microscope . The total number of colonies (>50 cells/colony) was counted, and the results are represented as the average of three independent experiments conducted over multiple days.0/G1, S and G2/M phases was determined using Cell Quest acquisition software .NOZ and GBC-SD cells were treated with various concentrations of cordycepin  for 48 h. Then, the cells were collected and washed twice with PBS. After fixing the cells in cold 70% ethanol at 4 °C overnight, the cells were washed and then resuspended in cold PBS and incubated with 10 mg/mL RNase and 1 mg/mL propidium iodide (PI)  at 37 °C for 30 min in the dark. The samples were analyzed by flow cytometry , and the percentage of cells in the G6 cells per well and treated with different concentrations of cordycepin  for 48 h. Then, the cells were harvested and washed twice with cold PBS and resuspended at a density of 1 × 106 cells/mL in 100 µL of binding buffer containing 5 µL of Annexin V-FITC and 1 μL of PI working solution (100 μg/mL). After incubation at room temperature for 15 min in the dark, 400 μL of binding buffer were added to each sample. Apoptosis was analyzed by flow cytometry  for at least 10,000 events.The analysis of apoptosis was performed according to the manufacturer’s instructions for the Annexin V/PI apoptosis kit . NOZ and GBC-SD cells were seeded in 6-well plates  with 1 × 10The ΔΨm was analyzed by fluorescence microscopy using the Rhodamine 123  probe. After treatment with various concentrations of cordycepin  for 48 h, the cells were collected and washed twice with cold PBS, followed by incubation with Rhodamine 123 at 37 °C for 30 min in the dark. After that, the cells were washed twice with cold PBS and analyzed by flow cytometry.After treatment with cordycepin  for 48 h, the cells were collected and lysed in RIPA buffer  supplemented with protease inhibitor . The total protein concentration of each sample was determined using the BCA protein assay  with BSA as a standard. Equal protein extracts (60 μg protein per lane) were separated by SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes . Then, the membranes were blocked with 5% skim milk and incubated with anti-caspase-3, anti-caspase-9, anti-Bcl-2, anti-Bax, anti-PARP, anti-Cyclin A, anti-CDK2, anti-21, anti-p27 and anti-β-actin antibodies  at 4 °C overnight, followed by incubation with a horseradish peroxidase-conjugated goat anti-rabbit/anti-mouse secondary antibody . Proteins were visualized using the Gel Doc 2000 .6) in log-phase growth were resuspended in 100 μL PBS and then injected into the left axilla of nude mice. On Day 5, these mice were randomly divided into three groups (6 mice/group). The first group received an intraperitoneally (i.p.) injection of William’s medium each day. The other two groups were administered an i.p. injection of cordycepin at 10 mg/kg and 50 mg/kg every day. On Day 22, all nude mice were sacrificed, and the tumor tissue was removed and weighed.Six- to eight-week-old BALB/c homozygous (nu/nu) nude mice (18–20 g body weight) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. . The mice were maintained in a specific pathogen-free environment. All procedures were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University and were carried out in accordance with the institutional guidelines of Shanghai Jiaotong University . NOZ cells  were considered statistically significant for all tests.Statistical analyses were conducted using SPSS 18.0 software. All assays were performed independently three times, and all quantified data are expressed as the mean ± SD or as indicated. Statistical significance was calculated using the Student’s Our study revealed that cordycepin suppressed the proliferation and induced the apoptosis of NOZ and GBC-SD cells via the activation of the mitochondrial-mediated intrinsic caspase pathway. These results indicate that cordycepin can have a significant anti-tumor effect in human gallbladder cancer cells, which suggests the potential application of cordycepin in the development of new anticancer drugs for the treatment of gallbladder cancer.


ATL1103 is a second-generation antisense oligomer targeting the human growth hormone (GH) receptor. This phase 2 randomised, open-label, parallel-group study assessed the potential of ATL1103 as a treatment for acromegaly.Twenty-six patients with active acromegaly  were randomised to subcutaneous ATL1103 200 mg either once or twice weekly for 13 weeks and monitored for a further 8-week washout period.The primary efficacy measures were change in IGF-I at week 14, compared to baseline and between cohorts. For secondary endpoints , GH, growth hormone-binding protein (GHBP)), comparison was between baseline and week 14. Safety was assessed by reported adverse events.P = 0.0002). Between cohort comparison at week 14 demonstrated the median fall in IGF-I to be 25.8% (P = 0.0012) greater with twice-weekly dosing. In the twice-weekly cohort, IGF-I was still declining at week 14, and remained lower at week 21 than at baseline by a median of 18.7% (P = 0.0005). Compared to baseline, by week 14, IGFBP3 and ALS had declined by a median of 8.9% (P = 0.027) and 16.7% (P = 0.017) with twice-weekly ATL1103; GH had increased by a median of 46% at week 14 (P = 0.001). IGFBP3, ALS and GH did not change with weekly ATL1103. GHBP fell by a median of 23.6% and 48.8% in the once- and twice-weekly cohorts (P = 0.027 and P = 0.005) respectively. ATL1103 was well tolerated, although 84.6% of patients experienced mild-to-moderate injection-site reactions. This study provides proof of concept that ATL1103 is able to significantly lower IGF-I in patients with acromegaly.Baseline median IGF-I was 447 and 649 ng/mL in the once- and twice-weekly groups respectively. Compared to baseline, at week 14, twice-weekly ATL1103 resulted in a median fall in IGF-I of 27.8% ( Acromegaly is a rare, chronic, life-shortening disease caused by hypersecretion of growth hormone (GH), virtually always due to a pituitary adenoma, that in turn results in elevated circulating levels of insulin-like growth factor 1 (IGF-I) . The GH Antisense oligonucleotides (ASOs) are single-stranded synthetic oligonucleotides that have been developed as therapeutic agents. Translation of mRNA, and hence, protein synthesis, is inhibited by sequence-specific ASOs that bind target pre-mRNA and/or mRNA . In the GHR) mRNA (https://www.asx.com.au/asxpdf/20111207/pdf/4234016x2cj5xn.pdf).ATL1103 is a second-generation, antisense oligomer designed to inhibit translation of human growth hormone receptor (HR) mRNA . It compHR) mRNA . Phase 1The objectives of this study were to evaluate the safety, tolerability and efficacy of ATL1103 in patients with acromegaly. Serum IGF-I was the primary measure of efficacy, with the other components of the IGF ternary complex, namely IGF-binding protein 3 (IGFBP3) and acid labile subunit (ALS) being additional measures of disease activity. Circulating GH and GHBP, the cleaved extracellular component of the GHR, were monitored to provide insight into the physiological implications of an ASO targeting the GHR. For the primary efficacy variable, the null hypothesis of no percentage change in fasting IGF-I levels from baseline to week 14 was tested for each treatment regimen.This was a phase 2 randomised, open-label, parallel-group study of the safety, tolerability, pharmacokinetics and efficacy of two subcutaneous dosing regimens of ATL1103 in patients with acromegaly .Figure 2Patients whoprovided written informed consent in accordance with local regulations,were 18–80 years of age, inclusive,had acromegaly due to pituitary adenoma (microadenoma or macroadenoma) identified by MRI,had serum IGF-I level at screening >1.3 times the upper limit of normal (ULN),had documented serum GH nadir levels >1 ng/mL at all test time points within the 2 h post oral glucose load for an oral glucose tolerance test (OGTT) ,were acromegaly treatment naïve or who had not taken other acromegaly medications for at least the following periods of time prior to IGF-I and GH screening tests: bromocriptine: 6 weeks; carbergoline: 8 weeks; quinagolide: 8 weeks; octreotide (subcutaneous): 4 weeks; pegvisomant: 8 weeks; octreotide LAR: 4 months; lanreotide : 4 months,2,had a BMI ≥19 kg/mhad adequate venous access to allow collection of multiple blood samples during the study,were female of non–child-bearing potential , or, if of child-bearing potential, agreed to use two approved methods of contraception for the duration of the study and for 3 months after administration of the last dose of study drug, or were male and surgically sterilised or agreed to use an approved method of contraception for the duration of the study and for 3 months after administration of the last dose of study drug,were willing and able to self-administer subcutaneous injections.Patients whohad acromegaly due to reasons other than pituitary adenoma,had a pituitary adenoma that was less than 3 mm distance from the optic chiasm,had undergone pituitary surgery within the 3 months preceding the screening visit,had received pituitary radiotherapy within the 1 year preceding the baseline visit,had insulin-treated diabetes or had commenced a new hypoglycaemic drug for diabetes within the 2 months prior to screening,had congestive heart failure, unstable angina, clinically significant cardiac arrhythmia, or a history of acute myocardial infarction within the 3 months preceding the baseline visit,had abnormal hepatic function at screening defined any of the following parameters >2× ULN: aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamyl transferase (GGT), alkaline phosphatase (ALP), prothrombin time or total bilirubin,had hepatitis B, hepatitis C or chronic liver disease,were pregnant or lactating,had known human immunodeficiency virus (HIV)  or history of immunodeficiency that may have compromised their safety or affected results from this study,had a history of alcohol or drug abuse in the 6-month period preceding the baseline visit.Patients were recruited in 13 tertiary referral centres in Australia, France, Spain and the United Kingdom.The study was approved by the NRES Committee East Midlands, Derby, UK; Bicêtre Hospital, France; Ethical Committee of Clinical Research, Hospital del al Santa Crue I Sant Pau, Barcelona, Spain and Royal Adelaide Human Research Ethics Committee, Adelaide, Australia. The study was registered as EudraCT 201200314730 and ANZCTR 12611000854932. Patients gave written informed consent.Patients received either ATL1103 200 mg once or twice weekly 3 and 4 days apart for 13 weeks, with every patient receiving three doses in the first week, administered every other day. Based on the tissue half-life of >4 weeks, experience from primate studies and data from the phase 1 study, additional ‘loading’ doses were administered in the first week.ATL1103 is formulated as a ‘ready-to-inject’ sterile solution at a concentration of 200 mg/mL, pH 7.4, in ‘Water for Injection’. Patients were taught to self-administer ATL1103 subcutaneously. After completion of drug administration at the end of 13 weeks, patients were monitored, off all therapy for acromegaly, for a further 8 weeks.All patients underwent pituitary MRI scans at baseline and at week 13 (completion of the study drug), which were independently reviewed by two ‘blinded’ expert pituitary neurosurgeons. Tumour diameter changes of 2 mm or more in any one dimension or tumour volume changes of more than 20% were considered significant.An OGTT with measurement of plasma glucose and serum GH was undertaken at baseline (after any drug washout) and again at the end of week 13. An adverse event (AE) assessment was undertaken at each of the 11 study visits from baseline until study conclusion.In addition to routine safety parameters, serum IGF-I, IGFBP3, ALS and GHBP were monitored. Ring size (fourth digit left hand) was measured using standard European-sized jewellers’ ring sets, and patients completed a signs and symptoms score  and the disease-generated ‘quality of life’ AcroQol. AcroQol comprises 22 questions divided into two main categories: physical and psychological function. The psychological category is further subdivided into appearance and personal relationships. Each question is scored out of 5, with a maximum score of 110 reflecting best possible quality of life. The result is then converted to a percentage .Permuted block randomisation (generated by a statistician and imported into the electronic case report form) was used to assign patients to either open-label, once- or twice-weekly ATL1003. Once initial data for a patient had been entered and the patient had fulfilled all inclusion criteria, a randomisation number and treatment regimen were generated. Treatment allocation was not known to the operational personnel until this randomisation was performed.Blocks of size four were used with no stratification for the first 24 patients. The list included an additional 24 randomisation numbers using a block size of 2  to allow for overage.Serum IGF-I, GH and IGFBP3 were measured centrally by IDS-iSYS  Ltd., Boldon, England, UK) assays at the Endocrine Laboratory, Universität München . Recombinant standards (98/574 for GH and 02/254 for IGF-I) yielded interassay variability of 4.0–8.7% (IGF-I) and 1.1–3.4% (GH) and sensitivity of 8.8 ng/mL (IGF-I) and 0.04 ng/mL (GH) . The limSerum ALS levels were measured in duplicate by sandwich immunometric assay using monoclonal antibodies directed against specific N- and C-terminal oligopeptides . A serumSerum GHBP levels were measured by a modification of the ligand immunofunctional assay with an in-house monoclonal anti-GHBP antibody. Within-assay CVs were 9.4% at 115 pmol/L and 6.1% at 1550 pmol/L. At the same concentrations, between-assay CVs were 8.5% and 10.9% respectively. The lower limit of quantification was 69 pmol/L, and the linear range was 69–3500 pmol/L. All samples from an individual subject were analysed in one run .The study was powered for within-group comparison of serum IGF-I (primary efficacy variable).s.d. of 30%. To achieve a level of significance of 0.05 with a two-sided test, it was determined that a minimum sample size of 12 patients per treatment was required to achieve a power of at least 80%.Based on a published pegvisomant study , a clinit test and Wilcoxon signed-rank test. In addition, a pre-specified, though not powered for between-treatment groups, comparison was performed using a Wilcoxon rank-sum test  for serum IGF-I 1 week after the last dose of study drug (week 14).The planned efficacy analyses were within the intention-to-treat group. For the primary efficacy variable, the null hypothesis of no percentage change in fasting IGF-I levels from baseline to week 14 was tested for each treatment group with a (two-sided) one-sample Baseline to week 14 testing for both cohorts was undertaken (Wilcoxon signed-rank test) for each of the secondary endpoints  and presented as median plus range.post hoc regression analysis of the relationship between change in IGF-I and dose/kg/week is reported with the associated 95% CI. Comparison of trapezoidal area under the curve for GH during the OGTTs is reported. Statistical significance is indicated by a P value <0.05.A Serum IGF-I data are expressed in mass units (ng/mL) and as a percentage of the upper limit of the age-related reference range.An independent Data Safety Monitoring Board (DSMB) was established prior to recruitment start, with an appropriate charter to direct decisions and monitor the trial safety results at intervals throughout the study and provided their recommendations as to whether the trial could continue as planned or whether there were any concerns. The DSMB comprised four individuals with appropriate experience in the areas of acromegaly, endocrine disorders, statistics and the conduct of clinical trials.Thirteen patients with active acromegaly (IGF-I >130% ULN at screening visit) (detailed in P = 0.0002), while no change was seen with once-weekly dosing. At week 14, the median fall in IGF-I was 25.8% greater with twice-weekly compared to once-weekly dosing (P = 0.0012). In the twice-weekly cohort, IGF-I levels were still declining at week 14, and at the end of the washout (week 21) remained lower than at baseline by a median of 18.7% (P = 0.0005)  (2.5 × ULN) and 649 ng/mL (239–831) (2.75 × ULN) ng/mL, in the once- and twice-weekly groups respectively. Compared to baseline, at week 14, ATL1103 at a dose of 200 mg twice weekly resulted in a median fall in serum IGF-I of 27.8%   and 5.Fi–975 .P = 0.0001) association between fall in IGF-I and the dose/kg/week ) demonstrated an estimated slope of regression of −8.27, indicative of a highly statistically significant  from baseline . Once-weekly ATL1103 did not result in a significant change in serum IGBP3.In the twice-weekly cohort, at week 14, there was a median fall in serum IGFBP3 of 8.9%  from baseline . Once-weekly ATL1103 did not result in a significant change in serum ALS.Compared to baseline, twice-weekly ATL1103 resulted in a median fall in ALS at week 14 of 16.7%  at week 14 compared to baseline (471 ng·min/mL (79–867)). There was no change in GH levels in the once-weekly cohort  in GHBP from baseline (525 pmol/L (<69–6434), while a median fall of 23.6%  was seen in the once-weekly cohort (1179 pmol/L (386–7637), P = 0.027) and was maintained through to week 21. In the twice-weekly cohort, the median fall in serum GHBP at week 21 was 40.4%  compared to baseline . Ring size was unchanged with once-weekly dosing.There was a statistically significant decrease in ring size circumference (mm) from baseline to week 13 for regimen 2, with a median decrease of −1.25 mm  vs 11 (7–30)). The median percentage fall from baseline at week 14 was greater for twice-weekly dosing 37.5% (range −185.7 to 91.7%) compared with 10.2% (range −33.3 to 83.3%) for once-weekly dosing, although the changes were not statistically significant.P = 0.002) and 3.4  respectively, but these parameters did not change significantly with twice-weekly ATL1103.The median absolute improvement in the physical dimension and global scores between baseline and week 14 in the once-weekly cohort were 6.25 . There was no significant improvement in the once-weekly cohort.In contrast, in the twice-weekly cohort, comparing baseline to week 14, the only significant finding was an improvement in the median absolute change for the appearance subsection of psychological dimension of 10.7  being the most common treatment-emergent AE, affecting 85% of patients in both cohorts . Four se9/L respectively; normal range 150 × 109/L), but these resolved either spontaneously or after treatment end (week 13). Two patients had elevated liver enzymes judged clinically significant: one patient taking the once-daily regimen had γ-glutamyltransferase, AST, ALT and ALP values above the normal limit (>ULN); one patient taking the twice-daily regimen had AST and ALT values >ULN. All effects on liver function were transient  profile was comparable for the two treatment groups . Almost Radiologically significant tumour diameter changes (2 mm or more in any one dimension or tumour volume changes of more than 20%) were reported in three patients. Tumour volume increased in two patients  and reduced in one patient on twice-weekly dosing (6.2 × 10.4 × 4.9 vs 2.6 × 5.7 × 4.1 mm). The changes were judged not to be clinically significant.The technology underpinning ASO therapy is rapidly advancing and has the potential to offer new therapeutic options across a broad spectrum of diseases. Disordered protein production or function is implicated in most pathological processes, and ‘gene silencing or activating’ by single-stranded ASOs against target RNA sequences is an attractive concept that permits greater specificity than can be achieved with small molecules or antibodies . The synThe data presented provide the ‘proof of concept’ that in patients with acromegaly, an ASO targeting the GHR can lower serum IGF-I and raise the prospect of a new and entirely novel therapy for acromegaly. Thirteen weeks of ATL1103 at a dose of 200 mg twice weekly lowered median serum IGF-I by 27.8%, with two 15%) of 13 patients achieving an IGF-I within the reference range. Serum IGF-I levels were still declining at week 14 and had not returned to baseline by the end of the 8-week (week 21) washout period , suggest% of 13 pIn conjunction with the data indicating a relationship between the dose/kg/week and the fall in serum IGF-I, it seems probable that larger doses of ATL1103 administered for longer are likely to result in a greater fall in IGF-I and offer the prospect of ‘normalisation’ of IGF-I in a greater proportion of patients. Reassuringly, the decline in serum IGF-I with twice-weekly treatment was paralleled by falls in the other elements of the IGF ternary complex, namely IGFBP3 and ALS.Circulating GHBP is the product of cleavage of the extracellular component of the GH receptor . In acroAs with pegvisomant, the reduction in serum IGF-I with twice-weekly ATL1103 was associated with a 46% increase in serum GH levels assessed during an OGTT. Studies with pegvisomant have demonstrated that the increased GH secretion is a consequence of negative feedback in response to the fall in circulating IGF-I induced by blocking GH action .It is encouraging that an improvement in the soft-tissue manifestations of acromegaly, indicated by the reduction in ring size, was seen with twice-weekly therapy. The short duration of therapy and small cohort size means it should not be a surprise that the fall in IGF-I was not associated with an improvement in SSS and only very modest improvements in quality of life as measured by AcroQol. Studies in larger numbers of patients treated for longer are required to demonstrate the impact of ATL1103 on well-being and quality of life.Almost all patients, approximately 85%, experienced ISRs, but otherwise ATL1103 was generally well tolerated with no apparent drug-related SAEs. There were four SAEs, of which three occurred in one patient, and all were judged as unlikely to be drug related; both patients completed the 13 weeks of therapy . ISRs arThere were no clinically significant changes in pituitary tumour size, but the short duration of treatment precludes meaningful conclusions about the long-term impact of ATL1103 on this parameter. Both the patients in whom tumour expansion of >2 mm was documented had discontinued somatostatin analogue therapy  prior to commencing ATL1103, raising the possibility of rebound expansion from somatostatin analogue-induced tumour shrinkage. Reassuringly, the experience from more than 10-year use of pegvisomant is that GHR-targeted therapy does not induce growth of pituitary adenomas .In summary, ATL1103 lowers IGF-I in acromegaly with biochemical changes consistent with downregulation of the GHR. As IGF-I was still declining at the end of the treatment period and with the knowledge that the dose/kg could be increased, ATL1103 has the potential to achieve disease control in a significant proportion of patients. Placebo-controlled studies of longer duration and using higher doses are needed to better assess the full potential of this novel treatment.PJT received research support from Antisense Therapeutics during the conduct of the study and with presenting the results. He has served on advisory boards (unpaid) and/or received research support from for Novartis, Ipsen, Chiasma, Strongbridge and Ono. J D C N-P served as a consultant and steering committee member and received research support from Novartis. P C received research support from Antisense Therapeutics, Novartis, Ipsen, Pfizer and Italfarmaco. T B received personal fees from Antisense Therapeutics and received research support and personal fees from Pfizer, Ipsen and Novartis. J A served as a speaker and on advisory boards for Pfizer, Ipsen and Novartis. G T and L A are employees of Antisense Therapeutics and own stock. M B received research support from Antisense Therapeutics, research support and personal fees from Chiasma and Novartis and personal fees from ONO. All other authors have no relevant disclosures.The study was funded by Antisense Therapeutics Limited .P J T contributed to the study design, identification of participating sites and review of study data and drafted the manuscript. J D C N-P participated in patient recruitment, contributed to drafting the manuscript and reviewed and approved the manuscript. J A, S J B A, A R, W D, P C, T B, S M W, C F, J A, A I M and D J T participated in patient recruitment and reviewed and approved the manuscript. G T and L A were involved in protocol development and reviewed and approved the manuscript. D R was responsible for the data analysis contained in the manuscript. M B contributed to the study design and measurement of IGF-I, GH, IGFBP3 and ALS and reviewed and approved the manuscript.


Ensifer meliloti strain RD64 and its parental strain 1021 were used to inoculate Medicago sativa plants. After verifying that the endogenous biosynthesis of IAA did not lead to genomic changes during the initial phases of the symbiotic process, we analyzed whether the overproduction of bacterial IAA inside root nodules influenced, in a coordinated manner, the activity of the nitrogen-fixing apparatus and the photosynthetic function, which are the two processes playing a key role in legume plant growth and productivity. Higher nitrogen-fixing activity and a greater amount of total nitrogen (N), carbon (C), Rubisco, nitrogen-rich amino acids, soluble sugars, and organic acids were measured for RD64-nodulated plants compared to the plants nodulated by the wild-type strain 1021. Furthermore, the RD64-nodulated plants showed a biomass increase over time, with the highest increment (more than 60%) being reached at six weeks after infection. Our findings show that the RD64-nodulated plants need more substrate derived from photosynthesis to generate the ATP required for their increased nitrogenase activity. This high carbohydrate demand further stimulates the photosynthetic function with the production of molecules that can be used to promote plant growth. We thus speculate that the use of PGPR able to stimulate both C and N metabolism with a balanced C/N ratio represents an efficient strategy to obtain substantial gains in plant productivity.Indole-3-acetic acid (IAA) is the main auxin acting as a phytohormone in many plant developmental processes. The ability to synthesize IAA is widely associated with plant growth-promoting rhizobacteria (PGPR). Several studies have been published on the potential application of PGPR to improve plant growth through the enhancement of their main metabolic processes. In this study, the IAA-overproducing  The extracts were dried with the vacuum at 30 °C in the dark and dissolved in phosphate buffer saline . An internal standard (10 nCi) of [3H]IAA (26 Ci/mmol)  was added before homogenization to estimate the recovery of extracted IAA during the work-up. The displacement immunoassay for IAA determination in leaves and nodulated roots was carried out using the methods described by Bianco and Defez ; Cb = [(27.43 × A648.6) – (8.12 × A664.2)]; (Ca+b) = [(5.24 × A664.2) + (22.24 × A648.6)]; (Cx+c) = [(1000A470 – 2.13Ca – 97.64Cb)/209] [Medicago leaves (0.1 g) at 42 DAI were collected and immediately frozen and ground in liquid NCb)/209] . The reshttp://www.walz.com/). Pulses (0.5 s) of red light (5000 mmol·m−2·s−1) were used to determine the maximum quantum yield of photosystem II (PSII), FV/FM (Fm-F0/Fm); the effective quantum yield of PSII, Y(II); and the non-photochemical quenching (NPQ)  and the light-induced NPQ generation during illumination with a red light intensity up to 1287 μmol·m4 plants C, thus i4 plants D compareMs-RD64 and Ms-1021 leaves were measured and compared over time, the following trend was observed: at 21 DAI, the total protein level per plant was lower in Ms-RD64; at 28 DAI, no significant difference was observed; and at 35 and 42 DAI, a significantly greater amount was measured for Ms-RD64 plants  and considered as the “response”. Ms-RD64 plants showed a higher level of sucrose compared to Ms-1021  being reached at 42 DAI , nitrogen-rich amino acids , and soluble proteins, including Rubisco, catalyzing the first major step of carbon fixation. The chlorophyll a fluorescence analysis revealed that the fluorescence emission per unit leaf area measured for Ms-RD64 and Ms-1021 plants did not change. These findings support the evidence that, in most species, substantial increases in crop yield do not correspond to significant changes in the rate of photosynthesis per unit leaf area [Ms-RD64 plants. The combination of higher non-photochemical quenching and a greater amount of carotenoids measured for these plants could prevent the likelihood of the formation of damaging free radicals. The over-investment in the Rubisco protein and nitrogen-rich amino acids observed for Ms-RD64 plants might have contributed to the more efficient stress response observed for these plants [Ms-RD64 plants was consistent with the higher level of storage compounds, such as poly-β-hydroxybutyrate, accumulated in the more extended interzone II-III of their root nodules [Ms-RD64 plants could be linked to the accumulation of such metabolic products. In legume plants, a substantial part of the energy input accumulated in the photosynthetic process is used to feed the energy-consuming nitrogen fixation. The overall data reported in this study show that the more efficient nitrogen-fixing apparatus of RD64-nodulted plants stimulates C assimilation through photosynthesis. The enhancement of these two processes occurred in a coordinated manner: the absolute levels of C and N significantly increased, but their relative ratios did not change when the Ms-1021 plants were used as a reference. The enhancement of plant growth promotion observed for RD64-nodulated plants could be related to this effect.In this study, the plant growth-promoting activities of the IAA-overproducing strain i et al. . Here, wanalyzed . The datanalyzed . The higeaf area ,54. In ce plants ,27,51. Ie plants ,56,57. T nodules . In addiIn recent years, several studies have shown that the use of PGPR represents an environmentally sound way of increasing the crop yield by stimulating plant growth through direct or indirect mechanisms. However, due to the properties often inconsistent with the inoculated PGPR, the use of PGPR in the agricultural industry is still limited. Considering that, in a sustainable agricultural system, crops need to overcome disease resistance, different abiotic stresses, and nutritional deficiency, an ideal PGPR should possess a broad spectrum of action. Here, we show that by using a simple system to increase the endogenous IAA biosynthesis in rhizobia strains, a PGPR with different capacities can be obtained. The results observed for Medicago plants nodulated by the IAA-overproducing strain RD64, lead us to speculate that PGPR stimulating the C and N metabolisms in a balanced manner could be used to significantly increase the plant yield.


The children engaged in club-based ball games (FC and OBG) had higher (p<0.05) lean body mass than NSC , performed better (p<0.05) in the YYIR1C test , and had lower (p<0.05) %HRmax after 1, 2, and 3 min of YYIR1C. Moreover, HRrest was lower (p<0.05) for FC than for OS and NSC (FC: 68 ± 9 vs OS: 72 ± 10 and NSC: 75 ± 10 bpm), and lower (p<0.05) for OBG than for NSC (OBG: 70 ± 10 vs NSC: 75 ± 10 bpm). This study found that 10–12-year-old Danish children engaged in club-based football and other ball games had better exercise capacity, lower resting heart rate, and higher muscle mass than children not engaged in leisure-time sports. Thus, participation in club-based leisure-time ball-game activities seems to be of importance for the fitness and health profile of prepubertal children.This study investigated whether the physical fitness and body composition of 10–12-year-old Danish children are related to participation in leisure-time club-based sporting activities. The study involved 544 Danish 10–12-year-old 5th-grade municipal schoolchildren . After answering a questionnaire about leisure-time sporting activities, the children were divided into four groups: football club participation , other ball games , other sports , and no sports-club participation . The children completed a battery of health and fitness tests, including a 20 m sprint test, a standing long-jump test, the Yo-Yo IR1 children's test (YYIR1C), and body composition, blood pressure, resting heart rate (HR It is known that precursors of adult cardiovascular disease (CVD) begin in childhood and that paediatric obesity, an important influence on overall CVD risk, tracks to later life and is associated with decreased quality of life and early mortality , 2. TherOrganised leisure-time sports-club participation is one potential way of increasing overall physical activity and fitness in young people, though other approaches can also be used, including physical education at school, school playground activities, and nonorganised leisure-time activities. Studies have shown that sports-club membership predicts higher levels of leisure-time physical activity , 8, and There are some indications that physical fitness and body composition in children are influenced positively by sports-club participation, at least in the case of certain sports, compared to age-matched children not participating in organised leisure-time sports-club activities. Several training studies have shown that short-term (6–26 weeks) team-sport training improves intermittent exercise performance and aerobic fitness  and elicThe aim of the present study was therefore to investigate whether the physical fitness and body composition of 10–12-year-old Danish boys and girls was related to the type of voluntary sports-club activity, i.e., participation in football, other ball games, and other sports versus school-children with no sports-club involvement.The study used a cross-sectional design, including physical testing and basal information, to test whether physical fitness and body composition are associated with a specific type of sports-club participation, e.g., football, other ball games, other sports, and no sports-club participation.Five hundred and forty-four Danish 10–12-year-old 5th-grade schoolchildren (269 boys and 275 girls) participated in the study. The children were aged 11.1 ± 0.4 years, were 150.5 ± 7.2 cm tall, and weighed 41.5 ± 8.4 kg. They came from nine different schools, five located in the capital regions of Frederiksberg and Copenhagen municipalities and four in the countryside regions of Frederikssund and Roskilde municipalities, about 40 km away from the capital.The study was approved by the Committees on Biomedical Research Ethics for the Capital Region of Denmark (J.no. H-15008117). Child assent and written informed parental consent were obtained for all participants. The children were tested during weeks 3–6 of the school year. All tests were performed by university staff members, supported by the schoolteachers.All the testing was performed in the late summer (August to September) at the beginning of 5th grade. The children answered two questions about their leisure-time physical activity: Do you do any sports in your spare time as a member of a sports club? If so, which? The children were divided into four different groups according to their leisure-time sports-club participation: football club (FC); other ball games ; other sports ; no sports-club participation (NSC). Ball games were defined as invasive ball games. rest) as well as performing several exercise tests . The tests were completed over two consecutive days with a requirement that no physical activity should be performed on the day before test day 1. Test day 1 included two 20 m sprints, two maximal horizontal jumps, and the YYIR1C. Before testing on test day 1, the children performed a standardised warm-up consisting of two sets of exercises 1 (forward running), 4 (circling partners), 12.1 (running 2 cones forward and 1 backwards), and 13 (high-speed running) from the FIFA 11+ warm-up programme [In addition to the questionnaire, the children underwent measurements of body composition , resting blood pressure, and resting heart rate  placed at 0 m (positioned 30 cm in front of the standing-start position) and at 20 m. The best time recorded was noted as the test result. The 20 m sprint has been shown to be a valid and reliable method for young children [The warm-up included instructions about completing the squat jump from the akimbo position. The children stood upright with their toes just behind a line with their feet parallel and shoulder-width apart; after flexing the knees to the squat position and holding the position for at least 2 s, the children jumped as far as they could and the distance from the start line to the heel position was measured. Using a measuring tape, the jump length was measured to the nearest centimetre. Each child had two attempts. If they failed to perform two correct jumps, they were allowed an additional attempt. The longest jump was noted as the test result. The maximal horizontal jump length test is reliable and has been shown to be strongly associated with lower- and upper-body maximal strength , 22.submax) was calculated as a mean over a 30 s period 15 s before to 15 s after 1, 2, and 3 min of exercise. HRmax was determined as the highest heart rate reached over a 15 s period during the YYIR1C test, as this test has been shown to be optimal for determining HRmax in 6-12-year-old children [The YYIR1C test was performed indoors on one half of a wooden-floor handball court in accordance with the studies by Ahler, Bendiksen, Krustrup & Wedderkopp  and Bendchildren , 15, 24.children –25. The children .rest was measured simultaneously in beats per minute(bpm) by a heart rate belt . The lowest value obtained over a 15 s period was noted as the HRrest value.Arterial blood pressure was measured with the subjects in the supine position following at least 10 min of rest in a quiet room providing an optimal setting for relaxation. Blood pressure was recorded in mmHg as the average of three measurements on the left upper arm using an automatic blood pressure monitor , with the cuff size adjusted to the arm as appropriate. HRHeight was measured with 0.1 cm precision using a Tanita Leicester transportable stadiometer . Body mass, body fat percentage, and lean body mass were measured using an InBody 230 multifrequency body composition analyser . The InBody 230 has been validated in 7–12-year-old children, showing precise lean body mass but underestimated fat mass and fat percentage .No maturation status assessment was performed. The subjects were weighed as described by Karelis, Chamberland, Aubertin-Leheudre, and Duval . Data ouPostural balance was assessed using a single-leg flamingo balance test . The chiData are reported as means (±SD). All data were tested for normality. Between-group differences were tested using one-way analyses of variance (ANOVA) in Sigma Plot 13.0 (SYSTAT). When a significant interaction was detected, a Bonferroni post hoc test was used to locate the significant differences. Significance was accepted at p<0.05. Some of the children did not attend in one of the two test days or did not want to complete some of the tests , which means that the number of participants may differ between each test. When differences are presented in percentages, they are calculated as ((xx-NSC)/NSC).rest than NSC children. FC also had a 4 bpm lower (p<0.05) HRrest than OS  HRrest than NSC boys  had a 7 bpm lower (p<0.05) HRNSC boys . There wNSC boys .Body composition variables related to height, weight, lean body mass, and fat percentage are presented in YYIR1C test performance was 57% and 41% better in FC and OBG than in NSC (p<0.05). FC performed 40% better than OS  submaximal HR measured as %HRthan NSC . FC and than NSC . FC had Children engaged in club-based football had better (p<0.05) 20 m sprint performance  than the three other groups, with FC boys having better (p<0.05) performance than the NSC boys. FC girls had 9% better (p<0.05) horizontal jump performance than NSC girls. There were no other significant between-group differences for horizontal jump performance and postural balance .The main findings of this study were that 10–12-year-old Danish children engaged in club-based leisure-time football and other ball games had better aerobic and muscular fitness than children not active in sports clubs. Furthermore, the children engaged in club-based football had better aerobic fitness than those engaged in other sports and were faster in 20 m sprinting compared to the other ball games. Most of these differences were present for girls as well as for boys, indicating that leisure-time ball-game activities are important for fitness and health profile in boys and girls aged 10–12 years. No significant differences were observed between groups, either overall or within gender, for fat percentage, BMI, horizontal jumping, and the flamingo balance test, except that those girls engaged in club-based football had higher horizontal jumping performance than girls in other sports or with no leisure-time sports.rest than NSC children. High HRrest is associated with increased risk of cardiovascular morbidity [rest for children engaged in ball-game activities was similar to values observed in 8–10-year-old Danish schoolchildren, though HRrest will decrease with increasing age [rest of 5-7 bpm between those involved in leisure-time ball-game activities and those with no sport club activity was also similar to the differences found between these groups for 8–10-year-olds [The present study showed that children engaged in leisure-time football and other ball games had 9% and 7% lower HRorbidity –32. An asing age , 33. Morear-olds , support2max=0.116xYYIR1C+42.3 ml/min/kg; [rest and better YYIR1C performance, provide further support for the notion that participation in leisure-time football is an activity that 10-12-year-old children can perform to improve aerobic fitness level.YYIR1C performance, which is known to be correlated to aerobic fitness in children , 24, was/min/kg; ), this d/min/kg; , 15 and /min/kg; . In the 2peak during treadmill testing to exhaustion [The differences in cardiovascular fitness could be related to the high amount of vigorous exercise carried out during ball games like football and handball , 13, 34 haustion .The present study also evaluated selected variables related to muscular fitness. The children playing football had 2–4% better 20 m sprint performance than the three other groups. One explanation for this could be the higher lean body mass, but the difference was only identified between FC and NSC, and those engaged in other team sports had poor sprinting performance despite high lean body mass values. Larsen et al.  observedThe findings of higher lean mass for football players and no significant difference in fat mass are in accordance with the Portuguese studies by Povóas et al. , 15 for A limitation of this study is the lack of information on the children's everyday activities . Obviously, it is also worth noting that the sports-club participation is voluntary and that the children may have chosen to take part in the sports-club activity based on skills, interests, and prior exposure to sports. The number of participants in the present study is high for FG, OS, and NSC, ranging from 141 to 197 participants, but not as high for the other ball games, with 42 participants, giving a stronger conclusion on sports-club football engagement compared to the other ball games. A further limitation is that there is no evaluation of the link between the number of years the children have been active in sports clubs and their fitness and health profile. Finally, it should be considered that the body composition analyser chosen for the study is well correlated with DXA scan method but is shown to underestimate fat mass and body fat percentage . This muIn conclusion, this study showed that 10–12-year-old children engaged in club-based ball games during leisure time had lower resting heart rate, better exercise capacity, and higher lean body mass than children not engaged in leisure-time sports. Thus, participation in club-based leisure-time ball-game activities seems to be of importance for the fitness and health profile of prepubertal children.


In this study we estimated the cost-effectiveness of adopting 3D Nonfluoroscopic Mapping Systems (NMSs) for catheter ablation (CA). This study includes patients who underwent supraventricular tachycardia (SVT) CA and atrial fibrillation (AF) CA from 2007 to 2016. A comparison was conducted between a reference year (2007) and the respective years for the two types of procedure in which the maximum optimization of patients' exposure using NMSs was obtained. We compared the data of all SVT CA performed solely using fluoroscopy in 2007 (Group I) and all SVT CA procedures performed using fluoroscopy together with an NMS in 2011 (Group II). There was also an important comparison made between AF CA procedures performed in 2007 (Group III) and AF CA in 2012 (Group IV), where patients' treatment in both years included the use of an NMS but where the software and hardware versions of the NMS were different. Two cost-effectiveness analyses were carried out. The first method was based on the alpha value (AV): the AV is a monetary reference value of avoided unit of exposure and is expressed as $/mansievert. The second one was based on the value of a statistical life (VSL): the VSL does not represent the cost value of a person's life, but the amount that a community would be willing to pay to reduce the risk of a person's death. The costs estimated from these two methods were compared to the real additional cost of using an NMS during that type of procedure in our EP Lab. The use of NMS reduced the effective dose of about 2.3 mSv for SVT and 23.8 mSv for AF CA procedures. The use of NMS, applying directly AV or VSL values, was not cost-effective for SVT CA for the most countries, whereas the use of an NMS during an AF CA seemed to be cost-effective for most of them. In our analysis the cost-effectiveness of the systematic use of NMSs strongly depended on the AV and VSL values considered. Nonetheless, the approach seemed to be cost-effective only during AF CA procedures. At the present time, the first-line of therapy for many cardiac arrhythmias is considered to be radiofrequency catheter ablation (CA) , 2. The In the last fifteen years 3D nonfluoroscopic mapping systems (NMSs) have become established as an important tool for CA of complex arrhythmias. Studies published in research literature have, in addition, demonstrated that NMSs permit a significant reduction in X-ray exposure during standard CA procedures –10. NeveEven with the reality of common practice in this area, the International Commission on Radiation Protection (ICRP) advises the adoption of the As Low As Reasonably Achievable (ALARA) concept in relation to the basic approach to the use of fluoroscopy in medicine .In an earlier paper, we have described that an extensive and daily use of NMSs in our context can drastically reduce X-ray exposure during CA procedures . In thisThis study is a retrospective analysis that involves consecutive patients who underwent supraventricular tachycardia (SVT) CA and atrial fibrillation (AF) CA procedures over 10 years of activity (from 2007 to 2016).In our previous paper, we reported the fluoroscopic data of these procedures focusing the analysis on the reduction of radiation exposure and we have shown that the decrease in X-ray exposure year after year was due to the systematic use of NMSs rather than other variables. This reduction of exposure has occurred without compromising efficacy, safety, and long-term positive outcomes of CA .In this study a comparison was conducted between a reference year (2007) and the respective years for the two types of procedure in which the maximum optimization of patients' exposure using NMSs was obtained. We compared the data of all SVT CA performed solely using fluoroscopy in 2007 (Group I) and all SVT CA procedures performed using fluoroscopy together with an NMS  in 2011 (Group II). There was also a second important comparison made between AF CA procedures performed in 2007 (Group III) and AF CA in 2012 (Group IV), where patients treatment in both years included the use of an NMS but where the software and hardware versions of the NMS were different .Prior to each procedure, we obtained the written informed consent of the patient. Baseline patient characteristics of the four groups are summarized in For this study, we compared fluoroscopy time and total X-ray exposure. Fluoroscopy time (FT) was defined as the cumulative duration of fluoroscopy during the entire procedure, whereas the patient radiation dose was assessed as the recorded dose-area product (DAP). χ2 test, while for the follow-up and long-term effects the Kaplan-Meier analysis of Disease-Free Survival and the Log-Rank Test were used. Statistical analyses were conducted by means of R software, version 3.4.3 , as proposed in the EHRA Consensus Document  instead of [$/man-mSv], since being related to a single patient exposure.The Value of a Statistical Life (VSL) based method aims at overcoming some limitations of the AV based method. It is based on the concept of the willingness to pay for specific level of risk reduction. The VSL does not represent the cost value of a person's life, but the amount that a community would be willing to pay to reduce the risk of one's death. The major advantage, with respect to the AV method, is that the economic quantification is performed after risk estimation and allows considering the age and the context of the population exposed to radiation . The risk assessment can be done using the coefficients reported in the recent (2011) report of the United States Environmental Protection Agency (EPA) EPA 402-R-11-001 ) in Group I to 1.03 Gy∗cm2 in Group II (95% CI [0.106-2.92]) (P<0.05). Similarly, the median ED during SVT CA procedures decreased from 2.54 mSv to 0.21 mSv, resulting in ED reduction ΔE equal to 2.33 mSv.In our EP Lab the use of an NMS was associated with reduced FT in Group II {median value 1.2 min, (95% CI [0.1-2.5]) (P<0.05)} compared to Group I {median value 16.6 min, (95% CI [12.2-21.8])}. There was a complementary reduction of the total X-ray exposure (DAP) from 12.70 GyThe reduction in X-ray exposure was obtained without prolonging the PT; there was a median value of 80 min (95% CI [70-108]) in Group I and a median value of 90 min (95% CI [72.5-106]) in Group II (P=NS). This occurred without any difference in the procedural outcome (P=NS) and achieved the same long-term effect in the follow-up (P=NS), as shown in ∗cm2 (95% CI [73.3-172]) in Group III to 18.9 Gy∗cm2 in Group IV (95% CI [14.8-23.4]) (P<0.05). Similarly, the median ED during AF CA procedures decreased from 27.6 mSv to 3.78 mSv resulting in a ED reduction (ΔE) equal to 23.82 mSv.In the case of our EP Lab, the use of an NMS resulted in reduced FT in Group IV {median value 7.5 min, (95% CI [6.0-9.7]) (P<0.05)} compared to Group III {median value 49.1 min, (95% CI [41.4-56.6])}. There was a complementary reduction of the total X-ray exposure (DAP) from 138 GyThe reduction in X-ray exposure was achieved without prolonging the PT; there was a median value of 240 min (95% CI [220-240]) in Group III and a median value of 186 min (95% CI [154-200]) in Group IV (P=NS). This happened without any difference in the procedural outcome and attained the same long-term outcome in the follow-up (P=NS), as shown in In this study we reported a reduction of 2.33 mSy using an NMS during SVT CA rather than SVT CA procedures performed with only fluoroscopy. Furthermore, we have also shown that the systematic use of an NMS, as well as the software and hardware updates of the NMS, has led to an ED reduction for each AF CA procedure equal to 23.82 mSv.Applying AV method the cost-effectiveness of the CA procedures is strongly related to the AV values adopted in each country. In For example for the Netherlands, applying equations (A), (C), and (D) will result in the following.∗AV = 2.33[mSv]∗619[$/mSv] = $1,442.3 (2014-USD)(A) Cost-Reduction = ΔE ∗High AV = ΔE∗1.50∗AV = 2.33[mSv]∗1.50∗619[$/mSv] = $2,163.4 (2014-USD)(C) High-Cost-Reduction = ΔE∗Low AV = ΔE∗0.50∗AV = 2.33[mSv]∗0.5∗619[$/mSv] = $721.1 (2014-USD)(D) Low-Cost-Reduction = ΔEIn contrast the systematic use of NMS during AF CA is economically advantageous for the majority of countries in For example for the Netherlands, applying equations (A), (C) and (D) will result in the following.∗AV = 23.82[mSv]∗619[$/mSv] = $14,744.58 (2014-USD)(A) Cost-Reduction = ΔE ∗High AV = ΔE∗1.50∗AV = 23.82[mSv]∗1.50∗619[$/mSv] = $22,116.87 (2014-USD)(C) High-Cost-Reduction = ΔE∗Low AV = ΔE∗0.50∗AV = 23.82[mSv]∗0.5∗619[$/mSv] = $7,372.29 (2014-USD)(D) Low-Cost-Reduction = ΔEApplying the VSL method the cost-effectiveness of the CA procedures is strongly related to the VSL values adopted in each country or organization and even in the same country; as for United States, there are different adopted VSL values across different agencies. In Considering OECD published values, inflated and exchanged to $2014-USD as reported in For example for the VLS proposed by Food and Drug Administration (FDA), applying equations (B), (E), and (F) will result in the following.∗VLS = ΔE∗LR∗VLS = 2.33[mSv]∗4.5[%/Sv]∗8.6[million$] = $901.7 (2014-USD)(B) Cost-Reduction = LARR∗HighVLS = ΔE∗LR∗1,50VLS = 2.33[mSv]∗4.5[%/Sv]∗1.50∗8.6[million$] = $1,353.6 (2014-USD)(E) High-Cost-Reduction = LARR∗LowVLS = ΔE∗LR∗0.50VLS = 2.33[mSv]∗4.5[%/Sv]∗0.50∗8.6[million$] = $450.9 (2014-USD)(F) Low-Cost-Reduction = LARRAgain if we considered only the AF CA procedure, the use of NMSs seems to be cost-effective.For example for the VLS proposed by Food and Drug Administration (FDA), applying equations (B), (E), and (F) will result in the following.∗VLS = ΔE∗LR∗VLS = 23.82[mSv]∗3.8[%/Sv]∗8.6[million$] = $7,784.4 (2014-USD)(B) Cost-Reduction = LARR∗HighVLS = ΔE∗LR∗1.50VLS = 23.82[mSv]∗3.8[%/Sv]∗1.50∗8.6[million$] = 11,676.6$ (2014-USD)(E) High-Cost-Reduction = LARR∗LowVLS = ΔE∗LR∗0.50VLS = 23.82[mSv]∗3.8[%/Sv]∗0.50∗8.6[million$] = 3,892.2$ (2014-USD)(F) Low-Cost-Reduction = LARRNowadays the efficacy of the use of NMSs for the treatment of complex and simple arrhythmias has been well-demonstrated , 23. An In our previous paper we have analysed the cost-effectiveness of the use of NMSs during paediatric CA procedures and we noted the positive effect of this working method in combination with the new attitude, but only if we consider the correction factor for children and the higher values of AV and VLS . This coIn this study two cost-benefit methods (AV and VLS) have been introduced and applied to determine if the investment of a systematic use of NMSs during CA procedures in adults is economically advantageous in our EP Lab. During the analysis we have found the same problem as in the paediatric population of our previous study: the two methods show a very large range of parameters and this range of values is connected not only to the fact that there are different countries where they are used, but also to the fact that, even within the same country, different values are given by various agencies. Considering these limitations, the main conclusion of our study is that there is an important difference between SVT CA procedures and AF CA procedures. In SVT patients the effective dose reduction is very low and therefore economically disadvantageous considering the majority of AV and VSL values. This is in contrast to what is published in literature using a different economic analysis . On the The number of patients in this study is low.The comparison between the third and fourth groups was not a comparison between AF CA procedures performed only with fluoroscopy and the same type of procedure performed with fluoroscopy plus an NMS. It can be assumed that without the use of the NMS the radiological exposure would have been greater in the third group, but this has not been demonstrated it in this study. We focused on comparison between the use of different software and hardware versions of NMS performing AF CA.Since the CA procedure is a complex procedure the conversion from DAP to the effective dose was done using common and accepted methods of conversion, which resulted in what is, in fact, only an estimation of effective dose reduction.Adopting the EPA's models for the quantification of cancer mortality risk, we did not consider the additional cost associated with not fatal cancers. Thus, the monetized benefit is underestimated.The costs of NMSs usage are not standardized among institutions; thus the results could not be applicable to other centers.In the authors' opinion the limitations of this study can be summarized as follows:Our study is a retrospective analysis and represents an economic evaluation of the systematic use of the NMSs during CA procedures in a real adult EP Lab context. The cost-effective advantageous of this use depends on which value of AV or VSL is applied. In our context the use of an NMS during SVT CA procedures results in being non-cost-effective considering the AV and VLS value of the most countries and agencies. Conversely, AF CA procedures with the use of a NMS seem to be cost-effective for most countries and agencies. This difference is related to the amount of dose reduction obtained during the procedure, which is very high during AF CA procedures. In our opinion this reduction is not only due to the use of NMS, but also due to the change in the mindset of the electrophysiologist. It is expected that the future decrease in unit cost of NMS will result in improved cost-effectiveness also for SVT CA procedures.


Radical gastrectomy with D2 lymphadenectomy is recognized as the standard treatment for resectable advanced gastric cancer. Preoperative fibrinogen and albumin measurements may bring clinical benefits in terms of providing advanced notice of a poor prognosis or recurrence in patients undergoing radical resection. The aim of this study was to identify markers that are predictive of a poor prognosis prior to surgery.  Eight hundred forty-two consecutive patients who underwent curative radical gastrectomy at our hospital between 2008 and 2012 were retrospectively reviewed. Based on plasma fibrinogen and serum albumin levels, preoperative fibrinogen and albumin scores (Fib-Alb scores) were investigated, and the prognostic significance was determined. n = 376), 1 (n = 327), or 2 (n = 139). When the correlation between the response rate and the change in the Fib-Alb score was investigated, the response rate was significantly lower in patients with an increased Fib-Alb score than in the other patients. In the survival analysis, patients in the Fib-Alb high-score group exhibited significantly worse recurrence-free survival (RFS) (P = 0.030) than patients in the other groups. A multivariate analysis using clinical stage and the change in the Fib-Alb score as covariates revealed that a change in the Fib-Alb score  was a significant independent predictive factor for RFS.  The patients were classified according to a Fib-Alb score of 0 ( The prognosis of patients with high fibrinogen and low albumin levels is poor. The Fib-Alb score was shown to be an independent prognostic factor for postoperative recurrence in gastric cancer patients who underwent radical gastrectomy. According to the latest data, gastric cancer (GC) is the sixth most common malignancy worldwide, with an estimated 1.2 million incident cases and 834,000 deaths occurring in 2016 . There aCancer Staging Manual of the Union for International Cancer Control/American Joint Committee on Cancer (UICC/AJCC). Informed consent was obtained from all the participants. This protocol was approved by the Ethics Committee of the First Hospital of Jilin University on February 19, 2013 (No. 2013-005). Neoadjuvant therapy may affect the levels of fibrinogen and albumin. Stage IV GC is considered difficult to resect via radical gastrectomy, which can affect patient survival. Thus, only patients who did not receive neoadjuvant therapy before gastrectomy and were not stage IV were enrolled in the analysis.Patients with histologically diagnosed GC who underwent gastrectomy with D2 lymphadenectomy at the Department of Gastric and Colorectal Surgery of the First Hospital of Jilin University between January 2008 and December 2012 were recruited for this study. Patients were excluded if they (1) underwent neoadjuvant therapy, (2) had previous malignancies or distant organ metastasis, (3) presented with positive margins, (4) were diagnosed with acute inflammatory disease, liver cirrhosis, chronic renal failure, autoimmune disease, or haematopoietic system disease, (5) died during the perioperative period or were lost to follow-up at 3 months after surgery, or (6) had stage IV GC. The TNM staging was determined based on the seventh edition of the Blood specimens were collected during the week before surgery. Plasma fibrinogen and serum albumin were measured immediately after blood sample collection. Plasma fibrinogen was assayed using Dade Thrombin Reagent  and a CA7000 analyser (Sysmex Corporation), and the cumulative coefficient of variation (CV) was 7.3%. Serum albumin was measured using the bromocresol green (BCG) dye method and a biochemical analyser , and the CV was 4.4% in the Clinical Laboratory of the First Hospital of Jilin University. Time-dependent receiver operating characteristic (ROC) analysis was performed to determine the optimal cut-off values of fibrinogen and albumin. Values above the optimal cut-off value were considered to be elevated, and those below the optimal cut-off value were considered to be decreased.To reflect the effects of the two factors and the combined impact on GC prognosis, a scoring system of 2, 1, or 0 was chosen. Fib-Alb scoring was performed as follows: patients with elevated fibrinogen and decreased albumin levels were assigned a Fib-Alb score of 2; those with only one of these abnormalities were assigned a Fib-Alb score of 1; and those with neither of these abnormalities were assigned a Fib-Alb score of 0.Follow-up was scheduled at three, six, and twelve months after surgery, and then every year thereafter until the death of the patients or the end of the study. Abdominal computed tomography (CT) was used to determine the clinical staging. CT scanning was performed every 6 months in 2 years and every 12 months in 2 to 5 years after surgery. Information on the general status and on postoperative therapy was collected during each follow-up. Survival time was defined as the duration of the time from the date of surgery to the date of death by any cause or to the date of the last successful interview if the patient was still living or was lost to follow-up.http://www.r-project.org). All P values were 2-tailed, and a P value < 0.05 was considered statistically significant.Continuous variables are described using the median (range), and categorical variables are described as the frequency (percentage). Optimal cut-off values of fibrinogen and albumin were calculated by time-dependent ROC curves using R language, where the difference between a true positive (TP) and false positive (FP) was maximal for the prediction of 5-year survival. Survival curves were generated using the Kaplan-Meier survival method and compared by the log-rank test. Cox proportional hazards regression models were used to obtain the hazard ratios (HRs) and 95% CIs for evaluating the influence of possible factors on GC survival. Multivariate stepwise Cox regression analysis was also performed to select variables that independently predicted GC survival. Statistical analyses were performed with SPSS version 18.0  and using R language  was the most common pathological type, followed by signet-ring cell carcinoma (8.9%) and others (5.7%). Only 30.6% of patients received 3 or more cycles of chemotherapy.The median levels of plasma fibrinogen and serum albumin among the patients were 3.11 (1.14-10.91) g/L and 37.2 (4.6-50.5) g/L, respectively. The optimal cut-off values for fibrinogen and albumin to best predict prognosis were obtained with time-dependent ROC curve analysis: the optimal cut-off value for fibrinogen was 3.39 g/L , and that for albumin was 34.9 g/L  Figures . Nearly P = 0.001) (P = 0.001) (P = 0.002) or 2  (P for trend < 0.001).The results of the univariate analysis are shown in = 0.001)  than the= 0.001)  compared< 0.001)  had a woP = 0.056, P = 0.088). The Fib-Alb score was shown to be an independent prognostic factor for patients with resectable GC (P for trend = 0.004); the HRs of a Fib-Alb score of 1 and a Fib-Alb score of 2 were 1.31 (95% CI: 1.03-1.66) and 1.61 (95% CI: 1.20-2.17), respectively. Moreover, older age, high TNM stage, positive vascular invasion, and positive neural invasion were also independent risk factors for overall survival.The multivariate analysis results of the factors are shown in P = 0.246). However, in the Cox regression model, this difference was not statistically significant (P = 0.111). In patients with TNM stage II-III disease, the results showed a trend that was consistent with that of the whole patient cohort; a higher Fib-Alb score was independently associated with worse survival   .P = 0.034) was higher in the patients with Fib-Alb scores of 2 than in those with Fib-Alb scores of 0 and 1, and this difference was statistically significant.A correlation analysis between the “fibrinogen”/“albumin”/“Fib-Alb score” and vascular invasion, pathological type, and histological grading was performed, and the results are shown in The univariate analysis indicated that older age (>60 years old), TNM stage II-III disease, moderate or high histological grade, positive vascular invasion, positive neural invasion, high fibrinogen levels (>3.39 g/L), low albumin levels (<34.9 g/L), and a Fib-Alb score of 1 and 2 were associated with a worse prognosis in GC patients compared to a score of 0. Moreover, the multivariate analysis showed that the Fib-Alb score but not the fibrinogen and albumin level was a powerful prognostic indicator for overall survival in TNM stage II-III patients.P = 0.028; Fib-Alb score = 2: HR, 1.61; 95% CI, 1.20-2.17; P = 0.001). In our study, a higher Fib-Alb score was found to be associated with a number of clinicopathological characteristics of GC patients, such as older age, vascular invasion, neural invasion, and TNM stage, which were independent risk factors for OS in GC, indicating that a higher Fib-Alb score might be associated with GC aggressiveness and progression. Studies have also demonstrated that high fibrinogen and low albumin levels could predict the prognosis of patients with several types of cancers [In this study, we used high fibrinogen (>3.39 g/L) or low albumin (<34.9 g/L) levels as prognostic indicators for GC patient survival. However, the combination of plasma fibrinogen and serum albumin levels was identified as a better predictor of prognosis ; however, the results for the Fib-Alb score were negative in the multivariate analysis. Positive results were only found for TNM stage II and III patients. These divergent results may be due to the weak influence of Fib-Alb; the impacts on the prognosis are not found when the number of analysed cases is low. When TNM stage II and III patients were combined and the number of cases increased, their correlation with prognosis was shown to be significant. Thus, we combined stage II and stage III in the analyses.P = 0.04) in the univariate analysis  and poorly differentiated histological grading (69.6%). The patients diagnosed with poorly differentiated histological types showed a poor 5-year survival rate compared to those with well or moderately differentiated histological types . This result may be due to inconsistencies in postoperative chemotherapy regimens and in the number of cycles. Another reason is that the number of cases is small; the survival results may change as the number of cases is increased. Zhang et al. reported that patients with TNM stage II-III disease and high preoperative fibrinogen/prealbumin ratio (FPR) values will benefit from neoadjuvant therapy [In our study, 258 patients  received 3 or more cycles of chemotherapy postoperatively. Although the 5-year survival rate showed an increasing trend, a significant difference was not found between the chemotherapy groups and nonchemotherapy groups in the univariate analysis (57.9% vs.54.4%,  therapy . They foIn our study, due to limited data availability, we did not discuss the effects of perioperative blood transfusions on prognosis. Previous studies have found that blood transfusions in the perioperative period affect the survival of GC patients. Squires et al. demonstrated that perioperative blood transfusion could reduce the recurrence-free and overall survival rates of GC patients . Kanda eIn conclusion, the preoperative Fib-Alb score was a powerful and significant independent prognostic indicator of advanced GC, especially in patients with stage II/III disease. However, the detailed mechanisms underlying how the Fib-Alb score affects GC should be examined in the future. Moreover, the prognostic value of the Fib-Alb score in advanced GC patients needs to be determined in a larger-scale prospective study.